1. Field of the Invention
This invention relates generally to the field of nucleic acid chemistry. More specifically, it relates to methods of controlling the fluorescence of fluorescently labeled oligonucleotides in solution using a DNA binding compound. Additionally, it relates to methods for detecting degradation of fluorescently labeled single-stranded oligonucleotides in solution. Additionally, the invention relates to methods for detecting nucleic acid sequences by hybridization with a complementary oligonucleotide probe.
2. Description of the Related
Nucleic acid detection using oligonucleotide probes has become a standard method for specific target detection. Numerous modifications of the method have been described. Generally, a DNA sample is immobilized on a solid support and then hybridized to a labeled target-specific probe (see, for example, Falkow et al., U.S. Pat. No. 4,358,535, incorporated herein by reference).
Several nucleic acid detection methods have been described which involve selective cleavage of oligonucleotide probes following formation of probe-target hybridization duplexes. Detection of cleaved probes indicates the occurrence of hybridization and, hence, the presence of target sequences. For example, Saiki et al., 1985, Biotechnology 3:1008-1012, incorporated heroin by reference, describe "oligomer restriction" detection methods, in which hybridization of the target-specific probe generates a restriction site which is then cleaved by the corresponding restriction enzyme. PCT Patent Publication No. WO 89/09284, incorporated herein by reference, describes methods in which RNA probes are used to detect DNA target sequences. RNA probes hybridized to DNA target are cleaved using RNaseH, which selectively cleaves RNA in RNA-DNA hybrid duplexes. U.S. Pat. No. 5,210,015, incorporated herein by reference, describes methods which use the 5' to 3' exonuclease activity of a nucleic acid polymerase to cleave probes hybridized to target sequences and thereby release labeled oligonucleotide fragments for detection. These methods require an additional oligonucleodde hybridized upstream of the probe hybridization site to act as a primer for the polymerase-mediated extension reaction. Probe cleavage occurs concomitant with primer extension.
The invention of the polymerase chain reaction (PCR), a process for amplifying nucleic acids, enabled the detection of nucleic acids with greatly increased sensitivity and specificity. Using PCR, segments of single copy genomic DNA can be selectively amplified to an easily detectable level prior to detection. PCR methods are disclosed in U.S. Pat. No. 4,683,202, incorporated herein by reference. PCR and methods for detecting PCR products using an oligonucleotide probe capable of hybridizing with the amplified target nucleic acid are described in U.S. Pat. No. 4,683,195, and European Patent Publication No. 237,362, both incorporated herein by reference.
Similar to the methods for detecting unamplified nucleic acid described above, methods for detecting amplification product have been described which involve selective cleavage of hybridization probes following formation of probe-target hybridization duplexes. Saiki et al., 1985, Science 230:1350-1353, incorporated herein by reference, describe the application of "oligomer restriction" to the detection of amplified product. U.S. Pat. No. 5,210,015 also describes the analysis of PCR amplification products using the 5' to 3' exonuclease activity of a nucleic acid polymerase to cleave labeled probes hybridized to target sequences (see also Holland et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:7276-7280, incorporated herein by reference). Probes that hybridize to a region of the target nucleic acid bounded by the amplification primers are incorporated into the amplification reaction mixture. Hybridized probes are cleaved by the 5' to 3' nuclease activity of the polymerase during primer extension. Detection of labeled fragments indicates the occurrence of both primer extension and probe hybridization, and, therefore, amplification of the specific target sequence.
A number of agents have been described for labeling nucleic acids, whether probe or target, for facilitating detection of target nucleic acid. Labels have been described that provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, and enzymatic activity and include, for example, fluorophores, chromophores, radioactive isotopes (particularly .sup.32 P and .sup.125 I), electron-dense reagents, enzymes, and ligands having specific binding partners. Labeling can be achieved by a number of means, such as chemical modification of a primer or probe to incorporate a label or the use of polymerizing agents to incorporate a modified nucleoside triphosphate into an extension product.
A variety of fluorescent DNA binding compounds are known. These include intercalating agents which bind non-covalently to the stacked bases of nucleic acids and display a change in fluorescence, either an increase or shift to a different wavelength, as a result. U.S. Pat. No. 4,582,789, incorporated herein by reference, describes several intercalating moieties including psoralens. Ethidium bromide (EtBr) is an intercalating compound that displays increased fluorescence when bound to double-stranded DNA rather than when in free solution (Sharp et al., 1973, Biochemistry 12:3055, incorporated herein by reference).. Although EtBr can be used to detect both single- and double-stranded nucleic acids, the affinity of EtBr for single-stranded nucleic acid is relatively low. EtBr is routinely used to non-specifically detect nucleic acids following gel electrophoresis. Following size fractionation on an appropriate gel matrix, for example, agarose or acrylamide, the gel is soaked in a dilute solution of EtBr. The DNA is then visualized by examining the gel under UV light (see Maniatis et al., 1982 eds., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory, incorporated herein by reference).
A homogeneous assay for PCR and concurrent PCR product detection based on the increased fluorescence that ethidium bromide (EtBr) and other DNA binding labels exhibit when bound to double-stranded DNA is described in Higuchi et al., 1992, Bio/Techniques 10:413-417; Higuchi et al., 1993, Bio/Techniques 11:1026-1030; and European Patent Publication Nos. 487,218 and 512,334, each incorporated herein by reference. The methods allow direct detection of the increase of double-stranded DNA during an amplification reaction, most significantly from the increase in amplified target. However, these methods detect only the total amount of double-stranded DNA in the reaction and do not distinguish specific nucleic acid sequences; assay specificity depends on the specificity of the amplification reaction.
The use of oligonucleotide probes labeled with interacting fluorescent labels in nucleic acid hybridization assays is described in Morrison, 1992, in Nonisotopic DNA Probe Techniques, Kricka, ed., Academic Press, Inc., San Diego, Calif., chapter 13; and Heller and Morrison, 1985, in Rapid Detection and Identification of Infections Agents, Academic Press, Inc., San Diego, Calif., pages 245-256; both incorporated herein by reference. The methods rely on the change in fluorescence that occurs when suitable fluorescent labels are brought into close proximity, described in the literature as fluorescence energy transfer (FET), fluorescence resonance energy transfer, nonradiative energy transfer, long-range energy transfer, dipole-coupled energy transfer, or Forster energy transfer. A number of suitable fluorescent labels are known in the art and commercially available from, for example, Molecular Probes (Eugene, Oreg.).
Morrison, 1992, supra, described FET-based assay formats in which interacting fluorescent labels are bound to separate oligonucleotides that are either brought together or separated by probe hybridization. These assay formats, which require two probes, are described as either non-competitive or competitive, depending on whether probe-probe hybridization competes with probe-target hybridization. In an alternative assay format, one fluorescent label is bound to the hybridization probe, and the second fluorescent label is brought into close proximity by intercalating into the double-stranded hybridization duplex. No significant interaction occurs between the intercalating label and the unhybridized probe in solution. Because the intercalating label can intercalate into any double-stranded nucleic acid, this format is practical only for the detection of single stranded target nucleic acid.
In one embodiment of the nucleic acid detection methods described in U.S. Pat. No. 5,210,015, described above, a probe is used which is labeled with interacting fluorescent labels in close proximity. The labels are attached to the probe separated by one or more nucleotides such that probe degradation during amplification separates the labels, thereby producing a detectable change in fluorescence. Such multiply-labeled probes are difficult and costly to synthesize.
Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill of the art, are fully explained fully in the literature. See, for example, Sambrook et al., 1985, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series, Methods in Enzymology (Academic Press, Inc.), all of which are incorporated herein by reference. All patents, patent applications, and publications mentioned herein, both supra and infra, are incorporated herein by reference.